Megadraconema Cornutum, a New Genus and Species from Korea

ZOOLOGICAL SCIENCE 28: 68–84 (2011) ¤ 2011 Zoological Society of Japan

Megadraconema cornutum, a New Genus and Species from Korea, with a Discussion of Its Classification and Relationships within the Family Draconematidae (Nematoda, Desmodorida) Based on Morphological and Molecular Characters

Hyun Soo Rho1,2, Wilfrida Decraemer2,3, Martin Vinther Sørensen4, Won Gi Min1, Jongwoo Jung5 and Won Kim6 *

1East Sea Research Institute, Korean Ocean Research & Development Institute, Uljin, 695-1, Korea 2Royal Belgian Institute of Natural Sciences, Department of Recent Invertebrates, Vautierstraat 29, B-1000 Brussels, Belgium 3Ghent University, Biology Department, Nematology Section, Ledeganckstraat 35, B-9000 Gent, Belgium 4Zoological Museum, The Natural History Museum of Denmark, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark 5Department of Science Education, Ewha Womans University, Seoul 120-750, Korea 6School of Biological Sciences, Seoul National University, Seoul 151-747, Korea

A new genus and species of Draconematidae Filipjev, 1918, Megadraconema cornutum gen. nov., sp. nov., inhabiting subtidal sediments in Jejudo, Korea is described. Megadraconema cornutum gen. nov., sp. nov. is mainly characterized by a long body (1630-2220 μm), presence of a transverse circle of well-developed papillae-like cuticular protrusions at the base of the lip region, a head capsule with reticular structure of subcuticle, an amphid with a pore-like opening, and an internal, bar-shaped fovea. The diagnosis of the family Draconematidae is emended and a key to genus is provided based on their major differential diagnostic characteristics, summarized in a table. Phy- logenetic relationships of all the genera within the Draconematidae are discussed for the first time, based on molecular analyses and morphological features. The phylogenetic position of the new genus and relationships within the family Draconematidae based on analysis of molecular sequence data are examined. Analysis of 18S rRNA gene sequences does not support the currently accepted classification, and indicates paraphyly of the subfamily Draconematinae.

Key words: morphology, morphometrics, key, SEM, DIC photomicrographs, 18S rRNA, taxonomy

also been found frequently on the surface of sea grasses, INTRODUCTION and in coralline habitats (Allen and Noffsinger, 1978; Draconematid nematodes are mainly characterized by the Decraemer et al., 1997; Karssen and van Aelst, 2002). presence of specialized locomotory structures, such as Decraemer et al. (1997) revised the family Draconemati- cephalic adhesion tubes (CAT) in the head region and sublat- dae Filipjev, 1918 and classified 14 genera of 70 valid spe- eral and subventral posterior adhesion tubes (PAT) in the pos- cies into two subfamilies: the Draconematinae Filipjev, 1918 terior body region (Allen and Noffsinger, 1978; Decraemer, including four genera of 41 species and Prochaetosomatinae 1989; Decraemer et al., 1997). Table 3 gives an overview of Allen and Noffsinger, 1978 including 10 genera of 29 spe- the diagnostic features within the Draconematidae. Dra- cies. Since this revision, 14 new draconematid species have conematid nematodes live in various marine habitats, from been described: Dinetia decraemerae Rho et al., 2006, the shallow intertidal sandy beaches to deep-sea hydrother- Dinetia orientalis Rho and Kim, 2005c, Dracogalerus mal vents, and are found throughout the world. They have koreanus Rho and Kim, 2005d, Paradraconema jejuense Rho and Kim, 2005, Prochaetosoma dokdoense Rho et al., * Corresponding author. Phone: +82-2-880-6695; 2010 and Tenuidraconema koreense Rho and Kim, 2004a Fax : +82-2-872-1993; were discovered from Korea in the Northwest Pacific. E-mail: [email protected] Tenuidraconema philippinense Rho and Kim, 2005a was doi:10.2108/zsj.28.68 discovered from the Philippines in the West Pacific. A New Genus of Draconematids from Korea 69

Tenuidraconema tongaense Rho et al., 2007 was discovered species of the Draconematidae have been hitherto recorded from the Tonga Islands in the Southwest Pacific. Cygnonema from various habitats in the Northwest Pacific Ocean (Table belgicae Raes et al., 2009a, Cygnonema verum Raes et al., 1). In our continuous investigation of the free-living nemato- 2009a and Tenuidraconema microsperma Raes et al., fauna in this region, which started in 2002, an unknown 2009b were discovered from the Porcupine Seabight in the draconematid species was collected from the washings of Northeast Atlantic. Dracognomus americanus Karssen and shallow subtidal detritus and coarse sediments of Munseom van Aelst, 2002 from Guadeloupe in the Caribbean Sea, and Islet off Jejudo, Korea. Morphological studies under differen- Draconema brasiliense Venekey, Lage and Fonséca- tial interference contrast (DIC) microscope and scanning Genevois, 2005 and Draconema fluminense Venekey et al., electron microscope (SEM) revealed that the species is 2005 were discovered from the Brazilian coast in the clearly different from all known species and genera of the Southwest Atlantic. This brings the total number of dracone- family. The present paper describes this species as a new matid species to 84 (Karssen and van Aelst, 2002; Raes et genus and species, Megadraconema cornutum gen. nov., al., 2009a, b; Rho and Kim, 2004a, 2005a, b, c, d; Venekey sp. nov. and discusses its classification and phylogenetic et al., 2005; Rho et al., 2006, 2007, 2010). relationships with other members of the family Draconema- Since the discovery of the first draconematid nematode, tidae, based on both morphological and molecular charac- Draconema cephalatum Cobb, 1913 from the Yellow Sea ters. We also present an emended diagnosis of the (Steiner, 1921; originally as Chaetosoma cephalatum, 15 Draconematidae and a key to all genera in the family.

Table 1. Draconematid nematodes discovered from the Northwest Pacific Ocean.

Species Reference Geography Habitat and ecology Family Draconematidae Filipjev, 1918 Subfamily Draconematinae Filipjev, 1918 Draconema cephalatum Cobb, 1913 Steiner (1921); the Yellow sea, Japan, base of algae with sand Allen & Noffsinger the Philippines (1978) Draconema japonicum Kito, 1976 Kito (1976); Japan, Korea Sea weeds (Sargassum community); Rho & Kim (2004b) intertidal and subtidal sediments and various algae Paradraconema floridense Allen & Noffsinger, 1978 Allen & Noffsinger Japan calcareous alga (Halimeda sp.) (1978) Paradraconema singaporense Allen & Noffsinger, 1978 Allen & Noffsinger Singapore a alga (Gracillaris sp.) (1978) Paradracoenema jejuense Rho & Kim, 2005b Rho & Kim (2005b) Korea shallow subtidal zone (25–35 m), coarse sediments Dracograllus filipjevi Allen & Noffsinger, 1978 Allen & Noffsinger Japan, Korea holdfasts of kelp; shallow littoral zone (1978); (3–5 m deep), coralline algae Rho & Kim (2006) Dracograllus gerlachi Allen & Noffsiner, 1978 Allen & Noffsinger Japan brown algae growing on rocks (1978) Tenuidraconema koreense Rho & Kim, 2004a Rho & Kim (2004a) Korea subtidal zone (150–250 m), coarse sediments and various invertebrates (hermit crabs, sponges and bryozoans) Tenuidraconema philippinense Rho & Kim, 2005a Rho & Kim (2005a) the Philippines shallow subtidal zone (45 m), coarse sediments Subfamily Prochaetosomatinae Allen & Noffsinger, 1978 Cephalochaetosoma pacificum Kito, 1983 Kito (1983) the Philippines deep-sea (5507–5587 m), sediments and fragments of a broken coconut with fibrous coat Bathychaetosoma uchidai Kito, 1983 Kito (1983) the Philippines deep-sea (5507–5587 m), sediments and fragments of a broken coconut with fibrous coat Dracogalerus koreanus Rho & Kim, 2005d Rho & Kim (2005d) Korea shallow subtidal zone (10–35 m), coarse sediments and various invertebrates (sponges and bryozoans) Dinetia orientalis Rho & Kim, 2005c Rho & Kim (2005c) Korea subtidal zone (250–300 m), sediments and small logs Dinetia decraemerae Rho et al., 2006 Rho et al. (2006) Korea subtidal zone (200–250 m), coarse sediments and small logs infested with limnorid isopods Prochaetosoma dokdoense Rho et al., 2010 Rho et al. (2010) Korea Coarse detritus and shell gravels 70 H. S. Rho et al.

drous glycerin following Seinhorst (1959) and mounted in anhy- MATERIALS AND METHODS drous glycerin on H-S slides to examine both sides of the speci- Sampling of taxa mens (Shirayama et al., 1993). All specimens used in this study, Draconematid nematodes studied in the present paper were except Megadraconema cornutum gen. nov., sp. nov., Draconema collected by SCUBA diver in various intertidal and shallow subtidal japonicum Kito, 1976 and Paradraconema jejuense Rho and Kim, habitats in Korean waters (Fig. 1); e.g., benthic detritus and coarse 2005, were identified only to the generic level due to the small num- sediments; calcareous algae and various invertebrates, such as ber of specimens. sponges (Callyspongia elegans and Cliona celata), polychaete tubes (Pomatoleios krausii and Serpula vermocularis), crustaceans Light and scanning electron microscopy (hermit crabs: Dardanus arrosor and Pagurus pectinatus), and The nematodes were observed with an Olympus BX51 micro- dead and living bryozoans (Adenonella platalea and scope equipped with DIC. All drawings and measurements were Thalamoporella lioticha). Deep-sea specimens of Prochaetosoma made with a drawing tube. Photographs were taken with a Nikon sp. 2 for molecular analyses were collected with a fishing net. Coolpix 990 digital camera, and the quality of the images was improved with Adobe Photoshop V7.0 software. Sample processing and preparation of specimens Two males, three females, and two fourth stage juveniles were Meiofaunal organisms extracted from samples through fresh- examined using a scanning electron microscope (HITACHI S-4100). ° water shocking (Kristensen and Higgins, 1989) were filtered through They were prefixed overnight at 4 C in a 2.5% phosphate buffered a 63 μm mesh-size sieve, and fixed in 4% buffered formalin in sea glutaraldehyde, followed by post fixation at 4°C with 1% phosphate water. They were kept in the formalin in seawater for morphological buffered osmium tetraoxide. After dehydration through a graded studies, or stored in 99% ethanol for molecular analyses. Coarse series of ethanol (40–100%, with 10% intervals) for 30 min each, the detritus and sediments were removed from the sample by decanta- specimens were critical-point dried and coated with gold-palladium tion, and the meiobenthos was extracted subsequently by flotation in a high vacuum evaporator. The images were scanned with a in Ludox® (DuPont) HS 40 (Burgess, 2001). The draconematid CanoScan LIDE 60, digitally improved with Adobe Photoshop v.7.0, nematodes were sorted out from the mixed meiobenthos under a and mounted with the help of Microsoft Office PowerPoint 2003. high magnification of LEICA MZ 8 stereomicroscope equipped with Type specimens are deposited in the nematode collections of DIC. Specimens for morphological studies were transferred to anhy- the Royal Belgian Institute of Natural Sciences, Brussels, Belgium (RBINS), the National Institute of Biological Resources, Incheon, Korea (NIBR), the Invertebrate Resources Bank of Korea, Seoul National University (IRBK), and the specimen conservation room of the Marine Environment Research Department, Korea Ocean Research and Development Institute (KORDI).

Terminology and abbreviations The terminology of the description and the measurements were conducted following Decraemer (1989). Abbreviations used in the text are as follows: L: total body length; CAT: cephalic adhesion tubes; CATn: number of cephalic adhesion tubes; mbd: maximum body diameter at mid body level; (mbd): minimum body diameter; mbd ph: maximum body diameter in pharyngeal region; ph: length of pharynx; PAT: posterior adhesion tubes; 1SlATl: length of anteriormost sublateral posterior adhesion tubes; SlATn: number of sublateral posterior adhesion tubes; 1SvATl: length of anteriormost subventral posterior adhesion tubes; SvATn: number of subventral posterior adhesion tubes; VAT: ventral adhesion tubes; t: tail length; tmr: length of non-annulated tail terminus; t/tmr: proportion of tail length to non-annulated tail terminus; abd: anal body diameter; spic: spicule or length of spicule measured along the median line; gub: gubernaculum or length of gubernaculums; V: position of the vulva as a percentage of the total body length from anterior; a, b, c, c′: proportions sensu de Man (1880).

DNA extraction, amplification, purification and sequencing To infer the molecular phylogenetic position and relationships of Megadraconema cornutum gen. nov., sp. nov. within the family Draconematidae, nearly complete 18S rRNA gene sequences of 10 draconematid nematodes (Draconema japonicum, Megadraconema cornutum gen. nov., sp. nov., Paradraconema sp., Paradraconema jejuense, Dracograllus sp. 1, Dracograllus sp. 2, Prochaetosoma sp. 1, Prochaetosoma sp. 2, Prochaetosoma sp. 3 and Prochaetosoma Fig. 1. Map showing sampling localities of the draconematid and sp. 4) and one epsilonematid nematode (Epsilonema sp.) were epsilonematid nematodes studied. (1) Munseom Islet amplified and sequenced (Table 2). A single animal for each of the (Megadraconema cornutum gen. nov., sp. nov., Dracograllus sp. 1, species was rinsed several times with distilled water to remove Prochaetosoma sp. 1 and Epsilonema sp.). (2) Beomseom Islet detritus particles. Samples were then subjected to DNA extraction. (Draconema japonicum and Prochaetosoma sp. 4). (3) Supseom Total genomic DNA used as a template for the polymerase chain Islet (Prochaetosoma sp. 3). (4) Seongsanpo (Paradraconema reaction (PCR) was extracted using a QIAamp tissue kit (QIAGEN jejuense). (5) Hyeobjae (Paradraconema sp.). (6) Geomundo Inc.), following the manufacturer’s instructions. (Dracograllus sp. 2). (7) Namae (Prochaetosoma sp. 2). The 18S rRNA gene of the draconematid nematodes was A New Genus of Draconematids from Korea 71

Table 2. Species used in molecular analyses, with GenBank accession numbers for sequences. Sequences reported in this paper are marked with an asterisk (*).

Taxonomy/taxa Locality/habitat/reference Accession number Ingroup Family Draconematidae Subfamily Draconematinae *Draconema japonicum Beomseom, Jejudo, Korea (33°13′21″N, 126°30′34″E); FJ182217 subtidal sediments (5 m deep) *Megadraconema cornutum gen. nov., sp. nov. Munseom Is., Jejudo, Korea (33°13′87″N, 126°34′04″E); FJ182219 subtidal detritus and coarse sediments with mollusk shell gravels and bryozoan particles (38.5 m deep) *Paradraconema sp. Hyeobjae, Jejudo, Korea (33°23′16″N, 126°14′42″E); FJ182221 intertidal brown algae *Paradraconema jejuense Seongsanpo, Jejudo, Korea (33°27′40″N, 126°56′33″E); FJ182220 subtidal sediments (30 m deep) *Dracograllus sp. 1 Munseom Is., Jejudo, Korea. (33°13′66″N, 126°34′18″E); FJ182215 subtidal detritus and shell gravels (37 m deep) *Dracograllus sp. 2 Geomundo Is., Jeonranamdo, Korea (34°05′57″N, 127°14′84″E); FJ182216 intertidal invertebrates Subfamily Prochaetosomatinae *Prochaetosoma sp. 1 Munseom Is., Jejudo, Korea (33°13′65″N, 126°34′61″E); FJ182222 subtidal detritus and shell gravels (34 m deep) *Prochaetosoma sp. 2 Namae, Gangwondo, Korea (37°57′07″N, 128°46′41″E); FJ182223 subtidal sediments and various invertebrates (200 m deep) *Prochaetosoma sp. 3 Supseom Is., Jejudo, Korea (33°13′71″N, 126°35′59″E); FJ182224 subtidal detritus and shell gravels (15–30 m deep) *Prochaetosoma sp. 4 Beomseom Is., Jejudo, Korea (33°13′95″N, 126°30′95″E); FJ182225 subtidal detritus and sands (37 m deep) Outgroup Family Epsilonematidae *Epsilonema sp. Munseom Is., Jejudo, Korea (33°13′87″N, 126°34′04″E); FJ182218 subtidal detritus and shell gravels (38.5 m deep) Epsilonematidae sp. Holterman et al. (2008) EF591340 amplified by PCR through the following procedure: an initial dena- the closely related family Epsilonematidae. Both forward and turation at 94°C for 3 min, followed by 30 cycles of denaturation at reverse strands were sequenced, and twelve nearly complete 18S 94°C for 1 min, primer annealing at 54.5°C for 1 min 30 sec and an rRNA gene sequences were initially aligned using the Muscle extension at 72°C for 2 min. Each reaction was terminated by a final ver.3.7 multiple alignment program (Edgar, 2004). The alignment extension step at 72°C for 10 min. All species were amplified with was further adjusted manually by eye and hand using Se-Al the primer set 328/329 designed on basis of sequences of the con- ver.2.0a11 (Rambaut, 1996). To evaluate the genetic distance of served regions at the 5′ and 3′ ends of eukaryotic homologs (Nelles the marine draconematid and epsilonematid nematodes, uncor- et al., 1984). Detailed information for PCR and the sequencing rected p-values of the 18S rRNA gene sequences were calculated primer sets used in the study is shown elsewhere (e.g., Table 3 in using MEGA ver.4.0 (Tamura et al., 2007) (Tables 6 and 7). Hwang et al., 2009). For each sample, 2.5 μl of genomic DNA was Maximum parsimony (MP), maximum-likelihood (ML), and amplified in a total reaction volume of 50 μl by using Taq DNA poly- Bayesian inference (BI) methods were used to reconstruct the phy- merase (Promega, Madison, USA), according to the manufacturer’s logeny from the aligned sequence data. The optimal sequence evo- recommendation, and then PCR products were isolated from 1% lution model was selected using MODELTEST 3.7 (Posada and agarose gel and purified with a GeneClean (Bio 101) Nal/glass- Crandall, 1998), and the HKY+I+G model was selected as the opti- powder Kit. The purified PCR products of the 18S rRNA gene were mal model for the aligned data set under the likelihood ratio test. electrophoresed on an ABI PRISM® 3100 automated DNA Analyzer The following are parameters related to the selected model: base using a Big Dye Terminator Cycle Sequencing Ready Kit (Applied frequencies of A, C, G and T were 0.2552, 0.2151, 0.2630 and Biosystems) according to the manufacturer’s instructions. Base cor- 0.2667, respectively; transition-transversion ratio (Ti/Tv) was rection was carried out using Sequence Navigator v.1.0.1 program. 1.9162; proportion of invariable sites was 0.5419; shape parameter All corrected sequences have been submitted to GenBank (Acces- of Gamma distribution was 0.7598. The selected model and param- sion numbers FJ182215 to FJ182225). eters were applied to the analyses of ML and BI. The MP and ML analyses were performed with PAUP* 4.0b10 (Phylogenetic Analy- Sequence alignment and phylogenetic analysis of 18S rRNA sis Using Parsimony) computer software (Swofford, 2002), and BI gene sequences was conducted using MrBAYES 3.1.2 (Ronquist and Huelsenbeck, GenBank accession numbers of the twelve nematode species 2003). Reliability of the nodes of the MP and ML tree was assessed sequenced for this study are listed in Table 2. Molecular sequences by the bootstrap procedures with 1,000 and 100 replications, have not previously been produced from any of the ingroup taxa, respectively. The following settings for BI were applied: number of comprising species of the family Draconematidae. The outgroup generations 100,000; sample frequency 100; number of chains 4; taxa, Epsilonema sp. sequenced for this study and an unidentified burnin 10,000. Epsilonematidae species gathered from NCBI, were chosen from 72 H. S. Rho et al.

Table 3. Diagnostic features of Megadraconema gen. nov., with other draconematid genera of the family (after Decraemer et al., 1997).

Characters Taxa Position Lumen wall Head capsule Body cuticle CAT PAT Amphid Buccal cavity Pharynx of CAT of end bulb Apenodraconema present presence typical posterior to typical spiral with a small cylindrical, not thickened of spine helmet dorsal tooth with posteriorly slightly swollen Bathychaetosoma present presence modified posterior to typical spiral with a dorsal cylindrical, moderately of spine helmet tooth and two with well thickened subventral teeth developed endbulb Cephalochaetosoma present smooth or modified posterior to typical spiral with a dorsal cylindrical, moderately presence helmet tooth and two with well thickened of spine subventral teeth developed endbulb Cygnonema present smooth typical posterior to typical spiral with a small cylindrical, not thickened helmet dorsal tooth posteriorly slightly swollen Dinetia absent lateral field modified posterior to typical spiral unarmed or cylindrical, moderately present helmet with a small with well thickened dorsal tooth developed endbulb Dracogalerus present smooth modified on helmet typical spiral narrow, cylindrical, not thickened unarmed with posteriorly slightly swollen Dracognomus present smooth modified on helmet modified reduced with a small with mid-corpus moderately inverted U dorsal tooth swelling and thickened shape endbulb Dracograllus present smooth or typical on helmet typical loop shaped narrow, dumbbell- not thickened lateral field unarmed shaped present Draconactus present smooth typical posterior to typical spiral with a small cylindrical, not thickened helmet dorsal tooth with posteriorly slightly swollen Draconema present higher size typical on helmet typical loop shaped narrow, dumbbell- not thickened of the first unarmed shaped (7–14) annules Megadraconema present reticular typical on helmet typical Pore-opening narrow, dumbbell- not thickened gen. nov. subcuticle with a unarmed shaped on helmet longitudinal tubular bar Notochaetosoma present smooth typical posterior to typical spiral narrow, cylindrical, not thickened helmet unarmed with posteriorly slightly swollen Paradraconema present presence typical on helmet typical loop shaped narrow, dumbbell- not thickened of spines unarmed shaped Prochaetosoma present presence typical posterior to typical spiral with a dorsal cylindrical, strongly of spines helmet tooth and with well thickened two subventral developed teeth endbulb Tenuidraconema present lateral field typical on helmet or typical spiral narrow, cylindrical, not thickened present posterior to unarmed with well helmet developed endbulb

Diagnosis (emended after Decraemer et al., 1997). RESULTS Desmodoroidea. Body short, S-shaped, usually with more or Systematics based on morphological observations less pronounced, enlarged pharyngeal and mid-body region. Body cuticle annulated except for head capsule and tail ter- Draconematidae Filipjev, 1918 minus, often with minute vacuoles. Annules usually smooth, A New Genus of Draconematids from Korea 73 rarely with spines, minute vacuoles/granules or a longitudi- internal longitudinal bar, located sublateral to dorsolateral on nal lateral field in mid-body region or tail region in adults and head capsule, when present, or near anterior head end. juvenile stages. Cephalic sensory organs in three circles: six Cephalic adhesion tubes (CAT or cephalic ambulatory anterior labial papillae, six short posterior labial setae and setae) present, located dorsally on head capsule, just pos- four cephalic setae, and with or without a transverse circle terior to it, or more posterior in cervical region, varying in of well-developed papillae-like cuticular protrusions near number but absent in the first juvenile stage. Somatic setae anterior border of head capsule. Head capsule present, arranged in eight longitudinal rows; four rows in tail region. except in Dinetia. Amphidial fovea spiralized to elongate Buccal cavity narrow to well-developed, usually armed with loop-shaped, rarely reduced with inverted U-shaped or an a dorsal tooth, with or without two smaller ventrosublateral

Table 4. Morphometrics of Megadraconema cornutum gen. nov., sp. nov. All measurements are in μm and in the form: mean ± standard deviation (range). (Abbreviations are explained in the section of materials and methods)

Male Female J4 J3 J2 Holotype Paratypes Allotype Paratypes Paratypes Paratypes Paratypes n –6–5543 L 1800 1919 ± 216 1840 1847 ± 51.7 1412 ± 178.4 957.5 ± 148.6 770 ± 60 (1630–2220) (1815–1910) (1140–1550) (850–1170) (710–830) a 28.1 30 ± 2.2 20.1 21.8 ± 2.2 24.3 ± 4.6 28.4 ± 2.8 25.9 ± 2.8 (28.1–33.9) (18.5–23.7) (16.7–28.9) (25–31.9) (22.7–27.7) b 14.9 14.5 ± 1.4 13.1 13 ± 1.1 11.7 ± 0.6 10.1 ± 0.9 9 ± 0.3 (13–16.7) (11.1–13.9) (11–12.6) (9.3–11) (8.8–9.4) c 15.1 15.7 ± 1.9 18.9 19.5 ± 1.2 15.2 ± 1.9 11.3 ± 1.1 9.9 ± 0.7 (13.5–18) (17.8–20.7) (12.5–16.8) (10.7–12.9) (9.1–10.4) c′ 4.4 4.1 ± 0.2 4 4.3 ± 0.1 4.7 ± 0.4 4.9 ± 0.2 4.5 ± 0.4 (3.7–4.7) (4.1–4.5) (4.0–5.1) (4.6–5.1) (4.2–4.9) V – – 62 62.8 ± 1.8 – – – (60–65) mbd 64 64.3 ± 9.8 91 85.4 ± 10.7 60.6 ± 18.4 34 ± 6.6 30 ± 4 (48–77) (78–103) (45–91) (27–41) (26–34) (mbd) 31 34.5 ± 3.1 34 37 ± 2.7 31.4 ± 3.9 23.8 ± 5.6 22.3 ± 1.5 (32–38) (33–40) (26–36) (19–30) (21–24) mbd ph 75 81.7 ± 3.4 79 81.8 ± 5.4 78.8 ± 3.4 59.0 ± 10.9 52.3 ± 2.5 (78–88) (75–90) (74–83) (49–73) (50–55) ph 121 132.7 ± 16.1 140 142.8 ± 11.0 121 ± 20.1 95.8 ± 20.1 85.3 ± 4.6 (105–152) (133–161) (94–138) (80–125) (80–88) abd 27 29.8 ± 2.1 24 22 ± 0.7 20 ± 1.9 17.5 ± 1.3 17.3 ± 1.2 (27–33) (21–23) (18–23) (16–19) (16–18) t 119 122.5 ± 5.6 97 95 ± 4.1 92.6 ± 1.8 84.5 ± 6.5 77.7 ± 2.5 (113–128) (91–102) (91–95) (78–91) (75–80) tmr 52 55.8 ± 1.7 54 55.6 ± 3.6 47 ± 1.6 44 ± 2.4 41.7 ± 1.2 (54–59) (52–60) (45–49) (42–47) (41–43) t/tmr 2.3 2.2 ± 0.1 1.8 1.7 ± 0.1 2.0 ± 0.1 1.9 ± 0.1 1.9 ± 0.1 (2–2.3) (1.6–1.8) (1.9–2.1) (1.9–2) (1.8–2) spic 102 112.5 ± 9.9––––– (109–130) gub 24 27.2 ± 2.1––––– (23–29) CATn 12 12 ± 0 12 12 ± 04 ± 03 ± 01 ± 0 (12–12) (12–12) (4–4) (3–3) (1–1) Amphid fovea length 13 13.3 ± 1.4 13 10.8 ± 0.6 8.4 ± 0.5 7.5 ± 0.6 7.3 ± 0.6 (11–15) (10.3–11.5) (8–9) (7–8) (7–8) 1SlATl 76 85.3 ± 6.7 76 79.2 ± 2 64.2 ± 3 56 ± 5.2 51.3 ± 8.6 (78–96) (77–81) (59–66) (52–63) (42–59) SlATn 13 14.8 ± 0.8 18 18 ± 0.7 6 ± 04 ± 02 ± 0 (14–16) (17–19) (6–6) (4–4) (2–2) 1SvATl 67 72.5 ± 3.5 71 72 ± 4.6 63.4 ± 1.8 – – (69–78) (69–80) (61–66) SvATn 18 18.8 ± 0.4 19 19.8 ± 0.4 10 ± 0– – (18–19) (19–20) (10–10) 74 H. S. Rho et al. teeth. Pharynx either dumbbell-shaped or largely cylindrical, four longitudinal rows: two sublateral and two subventral with or without well-defined terminal bulb (with medium rows with almost equally large number (13–20) of adhesion swelling in Dracognomus). Secretory-excretory system tubes. PAT becoming slightly shorter caudally. absent. At least the anteriormost posterior adhesion tubes Etymology. The generic name Megadraconema, neu- (PAT or posterior ambulatory setae) arranged in four longi- ter in gender, is a compound of “mega” (Gr., meaning large) tudinal rows: two sublateral and two subventral rows (except and the name of the type genus Draconema of the family in Prochaetosoma vitielloi Allen and Noffsinger, 1978 with Draconematidae. posteriorly a single ventral row) located on posterior third of body; PAT with or without differentiated (= bell- shaped) tip, usually straight, rarely long and flexible. Female reproductive system didelphic-amphidelphic, located anterior to PAT region. Male monorchic, single testis outstretched; copulatory apparatus with two spicules and a trough-shaped gubernaculum. Three caudal glands usually extending beyond anal region.

Draconematinae Filipjev, 1918 Genus Megadraconema gen. nov. Diagnosis. Draconemati- dae. Body typical Draconema- shaped, with strongly swollen pharyngeal region. Head capsule with thin-walled anterior collar-like portion and thickened main part with reticular subcuticle. Twelve CAT located dorsally on head capsule and arranged in two transverse rows composed of six longitudinal rows each. Amphid without sexual dimorphism in shape: a pore-like opening and an inner longitudinal tubular fovea located laterodorsally on head capsule. Amphid in fourth- and third-stage juveniles similar to adults; second-stage juveniles with pore-like opening connected with an inner elongated spiral fovea. A transverse circle of well-developed papillae-like cuticular protrusions present near anterior end of head capsule in all stages; number of protrusions variable among stages. Fig. 2. Megadraconema cornutum gen. nov., sp. nov., male (holotype RIT751, A–D; paratype, All PAT with well marked RIT753, E). (A) Habitus, lateral view. (B) Surface view of lateral head and pharyngeal region showing bell-shaped end and tongue- amphidial fovea and cephalic adhesion tubes. (C) Copulatory apparatus, lateral view. (D) Posterior tail like inner structure; situated region, lateral view. (E) Detail of head capsule showing lip region and amphidial fovea, lateral view. precloacally. PAT arranged in Scale bars: 50 μm (A), 20 μm (B–D), 10 μm (E). A New Genus of Draconematids from Korea 75

Type species. Megadraconema cornutum sp. nov. cuticular protrusions near the anterior border of the head Differential diagnosis. As shown in “Relationships capsule. within the family based on morphological features” of the Diagnosis. Body 1630–2220 μm long, with swollen pha- Discussion and Table 3, Megadraconema gen., nov. differs ryngeal region, 6–9% of total body length. Twelve CAT from all other genera of the Draconematidae by the pres- located anterodorsally on head capsule and arranged in two ence of a transverse circle of well-developed papillae-like transverse rows composed of six longitudinal rows each. cuticular protrusions at the base of the lip region, a head Amphid with pore-like opening, connected with short narrow capsule with reticular structure of subcuticle, a pore-like inner longitudinal tubular fovea in both sexes and fourth and amphid opening, and an internal, bar-shaped amphidial third stage juveniles, located laterodorsally near anterior fovea. edge of head capsule. Second stage-juvenile with pore-like opening connected with an inner elongate spiral fovea. All Megadraconema cornutum gen. nov., sp. nov. PAT precloacal, arranged in four longitudinal rows: two sub- (Figs. 2–9) lateral rows with 13–16 adhesion tubes in males and 17–19 Type material. Holotype: adult male (RIT751). Allotype: adhesion tubes in females, and two subventral rows with adult female (RIT752). Paratypes: each set of five speci- 18–19 adhesion tubes in male and 19–20 adhesion tubes in mens (one male, one female, one fourth stage juvenile, one female. Spicule length up to 5.7% of total body length (102– third stage juvenile and one second stage juvenile: RIT753– 114 μm), slightly arcuate with well-developed velum; capitu- 757; NIBRIV0000105001, 0000105061–0000105064; lum of retracted spicule reaching to twelfth sublateral adhe- IRBK102–106) and 11 paratypes (four male, three female sion tube. Tail short, gradually tapering to non-annulated and two fourth stage juvenile, one third stage juvenile and conical tip (ratio c: 15.6 ± 1.7 in male and 19.4 ± 1.1 in one second stage juvenile: KORDI015–025). female). PAT becoming slightly shorter caudally. Etymology. The proposed specific name, cornutum, Type locality and habitat. A subtidal zone of Munseom refers to the transverse circle of well-developed papillae-like Islet off Jejudo, Korea (33°13′87″N, 126°34′04″E), collected from detritus and coarse sediments containing dead mollusk

Fig. 4. Megadraconema cornutum gen. nov., sp. nov., SEM photomicrographs of male (paratype, KORDI023). (A) Head region, en Fig. 3. Megadraconema cornutum gen. nov., sp. nov., DIC photo- face view, with indication of cephalic adhesion tubes (CAT) and a micrographs of male (paratype specimens KORDI015, A, C–D) and transverse circle of papillae-like cuticular protrusions (P). (B) Head female (paratype KORDI018, B). (A) Habitus, male, lateral view. (B) region, lateral view showing CAT, arrow points to amphidial opening Habitus, female, lateral view. (C) Head region, lateral view, with indi- (AF). (C) En face view of head with protruding lips and a transverse cation of cephalic adhesion tubes (CAT) and amphidial fovea (AF). circle of papillae-like cuticular protrusions. (D) Head region showing (D) Copulatory apparatus, lateral view. Scale bars: 100 μm (A, B), widened base of CAT, dorsal view. Scale bars: 25 μm (A, B), 15 μm 30 μm (C, D). (C, D). 76 H. S. Rho et al. shell gravel and bryozoan particles (25–38.5 m deep) on 7 2E). Twelve CAT located on anterior-third of thickened head Nov 2000, 13 Oct 2002, 23 Jun 2003 and 28 Oct 2005 by capsule and arranged in two transverse rows of six longitu- HS Rho and JW Choi. dinal pairs of tubular setae with enlarged insertion base and Measurements. See Table 4. slightly swollen tip (Fig. 4D); CAT bent ventrally, 36–40 μm Description. Males (mainly based on the holotype and in length, posterior tubes longer than anterior ones in holo- partly paratypes RIT753, KORDI015, KORDI023). Body with type. Four pairs of CAT located almost dorsally, and two swollen pharyngeal region (6–8% of total body length), nar- pairs of outer ones located laterodorsally (Fig. 4A, D). row and slightly widening part along anterior intestine, wider cylindrical part with reproductive system, and tail tapered posteriorly and ending in conical tip; anterior body often strongly ventrally curved in fixed specimens (Figs. 2A, 3A). Cuticle finely annulated except for head capsule and tail terminus, with no lateral differentiation. Head with well-cuticular- ized capsule. Head capsule truncated, dome-shaped (Fig. 2B, E) with thickened cuticle except for thinner anterior cuticular collar with fringed border; anterior border of capsule with a transverse circle of papillae-like cuticular protrusions (Figs. 2B, E, 4A, C). Surface of thick head capsule appearing smooth but with reticulated subcuticle (Figs. 2B, 3C); surface of head capsule slightly wrinkled in SEM (Fig. 4A–D). Lip region extruded, 5 μm in height, in holotype; lip region partially or completely retracted in most fixed specimens. Six lips well- developed: each lip with a short labial seta (8 μm long in holotype) (Fig. 2B, E). Ante- rior thin collar-like part of head capsule with about 18 setae in holotype, more or less arranged in two transverse rows; posterior row (about 12 setae in holotype) just in front of papillae-like cuticular protrusions; cephalic setae probably four, but indis- tinguishable from many sub- cephalic setae of differing size. Amphidial opening pore- like (2.4 μm in diameter, holotype) with protruding corpus gelatum in SEM (Fig. 4B), connected with an inner longi- Fig. 5. Megadraconema cornutum gen. nov., sp. nov., female (allotype RIT752). (A) Habitus, lateral tudinal tubular fovea (11 μm view. (B) Surface view of lateral head region. (C) Pharyngeal region, optical section. (D) Posterior sub- long or 19.6% of head cap- lateral (left side) and subventral adhesion tube (right side), lateral view. (E) Posterior tail region, lateral sule length in holotype) (Fig. view. (F) Detail of vulva and vagina, lateral view. Scale bars: 50 μm (A), 20 μm (B–F). A New Genus of Draconematids from Korea 77

Stoma narrow, unarmed. Pharynx dumbbell-shaped, lumen wall not thickened (Fig. 2A). Nerve ring at level of indentation of mid pharynx. Cardia well-developed (7.3 μm long in holotype). Intestine broad, more or less cylindrical. Secretory-excretory system not observed. Somatic setae hair-like with broadened base, 5–71 μm long in holotype; longest setae (mean length 56 μm) occurred at swollen anterior part. Somatic setae densely dis- tributed and arranged in 14 longitudinal rows in pharyngeal region: two rows each in mediodorsal, subdorsal, mediolat- eral, lateroventral, subventral, and medioventral positions (Fig. 2B). Slender region posterior to pharyngeal bulb and region anterior to PAT with eight longitudinal rows: two mediodorsal rows, two laterodorsal rows, two lateroventral rows, and two medioventral rows of somatic setae (Fig. 2A). Six pairs of pericloacal setae, three pre-cloacal pairs, two cloacal pairs and one post-cloacal pair, all uniformly tapered. Three pre-cloacal setae at each side composed of one short (10 μm long in holotype) and two long setae (anterior seta 21 μm long and posterior one 31 μm long, respectively, in holotype). Two cloacal setae at each side: anterior seta (30 μm long) longer than posterior one (16 μm long); single post-anal seta at each side 20 μm long, situated just posterior to cloacal opening in holotype. All PAT with well- marked bell-shaped tip containing tongue-like extension of inner canal, widened at insertion (Fig. 5D). PAT located anterior to cloacal opening and arranged in four longitudinal rows: two sublateral rows each with 13 adhesion tubes inter- Fig. 6. Megadraconema cornutum gen. nov., sp. nov., SEM photo- mingled with four somatic setae, and two subventral rows micrographs of female paratype KORDI024, A–C) and male (para- consisting of 18 (left side) and 17 (right side) adhesion type KORDI023, D). (A) Detail of head region showing amphidial tubes, respectively, without intermingling somatic setae in fovea and some papillae-like cuticular protrusions. (B) Amphid open- holotype. PAT becoming slightly shorter caudally. In holo- ing, lateral view. (C) Anal flap and cloacal region, oblique view. (D) type, first SlAT and SvAT located at 77% and 78.4% of body Non-annulated tail terminus. AF = Amphidial fovea; P = Papillae-like length, respectively. Distance between anteriormost and cuticular protrusions. Scale bars: 5 μm (A, C), 2 μm (B), 10 μm (D). posteriormost SlAT 10.6% of total body length in holotype. Reproductive system typical for Draconematidae: a single, outstretched anterior testis (monorchic) extending far tion of subcuticule somewhat different (Fig. 5B). Amphidial anteriorly up to 42.2% of total body length from anterior end pore-like opening 2 μm in diameter, inner tubular fovea and located ventrally to intestine in holotype (Fig. 2A). Spic- 13 μm long and 15.8% of head capsule length (Fig. 6A, B). ules slightly arcuate, 102–114 μm long, up to 5.7% of total Twelve CAT located on anterior third of head capsule, as in body length in holotype; gradually narrower posteriorly and male (Fig. 5B, C). All PAT located anterior to anus, arranged ventrally with well-developed velum; capitulum offset, usu- in four longitudinal rows; two sublateral rows each consisting ally blunt beak-shaped (Figs. 2C, 3D). Capitulum of of 18 adhesion tubes, and two subventral rows consisting of retracted spicule reaching up to twelfth sublateral adhesion 19 (left side) and 17 (right side) adhesion tubes, respec- tube. Gubernaculum thin, parallel to spicules. Short anal flap tively, in allotype; PAT without intermingling somatic setae. present and smooth. Anteriormost SlAT and SvAT located at 79.5% and 80.4% of Tail gradually tapering up to non-annulated conical tip, body length, respectively. Distance between anterior and 43.7% of tail length; three pairs of somatic setae, two sub- posterior SlAT 11.9% of total body length. dorsal and one subventral, on annulated part, and five pairs Reproductive system typical for Draconematidae, of setae, three subdorsal (long anterior and two short pos- didelphic-amphidelphic with ovaries reflexed to left side. terior pairs) and two subventral (anterior 37 μm and poste- Spermathecae not observed. Vagina long, pars vaginae rior 11 μm long) (Figs. 2D, 6D). Cuticle of non-annulated tail distalis surrounded by constrictor muscle. Vulval lips terminus finely vacuolated, mainly in dorsal region. Caudal protruding (Fig. 5F). Two pairs of paravulval setae present gland reaching to posterior fourth sublateral adhesion tube (19 μm long). Short anal flap present, not crenate (Fig. 6C). and ending on well-developed spinneret. Tail very short, gradually tapering up to conical, smooth Females (allotype RIT752 and paratypes KORDI018 terminus; terminus non-annulated but finely vacuolated dor- and KORDI024). Similar to male in most respects (Figs. 3B, sally, with well-developed spinneret, 55.7% of total tail 5A–D). Body with strongly enlarged pharyngeal region, 7– length (Fig. 5E). Tail with two pairs of somatic setae: one 9% of total body length; greatest body width at level of vulva pair subdorsally and one pair subventrally on annulated part, region; anterior body region slightly curved ventrally. Head and four pairs of somatic setae on smooth terminal part, i.e. region largely similar to that in male, but pattern of reticula- three subdorsal pairs and one subventral pair. Cuticle of 78 H. S. Rho et al.

non-annulated tail end finely vacuolated dorsally; well- region, 8.6% of total body length. Head capsule with a trans- developed spinneret present. verse circle of papillae-like protrusions as in adult; number Juveniles. Three juvenile stages (J2, J3, J4) were of protrusions lower than in adults (Figs. 7B, C, 8D). observed. Developmental stages were mainly differentiated Amphidial opening 1 μm in diameter; inner longitudinal fovea by the number of CAT and the arrangement (number of lon- 8 μm long and 24% of head capsule length. Four CAT gitudinal rows) and number of PAT. located about medially of thickened head capsule and Fourth-stage juvenile (RIT755). Habitus similar to adult arranged in one transverse row of tubular setae with (Figs. 7A, 8A). Body with strongly swollen pharyngeal enlarged insertion base and slightly swollen tip: inner two CAT situated dorsally, outer two laterodorsally. PAT arranged in three longitudinal rows anterior to anal opening, two sublateral rows, each consisting of six adhesion tubes and one row of VAT with 10 tubes. No intermingling somatic setae. Anteriormost SlAT and SvAT located at 76.5% of body length. Distance between anteriormost and posteriormost SlAT 11.3% of total body length. Genital primordium, 106 μm in length. Short anal flap present, not crenate. Tail gradually tapering up to conical, non-annulated but finely punctated end, 48.4% of total tail length (Fig. 7D). Annulated part of tail with one pair of subdorsal and one pair of subventral somatic setae; smooth part of tail with three pairs of somatic setae. Spinneret well-developed. Moulting third-stage juvenile (paratype RIT756). Habitus similar to adults (Figs. 8B, 9A). Pharyn- geal region strong swelling, 10.6% of total body length. Head capsule with slightly wrinkled surface and a transverse circles of papillae-like protrusions as in adults (Fig. 9B); number of protrusions smaller than in adults and fourth stage juveniles. Amphid similar to fourth stage juveniles, longitudinal tubular bar 23.8% of head capsule. Three CAT arranged in a transverse row about mid-way head capsule, one mediodorsal and two laterodorsal. PAT arranged in two sublateral longitudinal rows anterior to anal opening, each row consisting of four adhesion tubes and no intermingling somatic setae. PAT becoming slightly shorter caudally. Anteriormost SlAT located at 75.5% of body length. Distance between anteriormost and posteriormost SlAT 9.4% of total body length. Genital primordium 41 μm Fig. 7. Megadraconema cornutum gen. nov., sp. nov., fourth-stage juvenile (paratype RIT755). in length. Tail with numerous fine (A) Habitus, lateral view. (B) Head region, external view. (C) Anterior pharyngeal region, optical transverse striae and well-devel- section. (D) Posterior tail region, lateral view. Scale bars: 50 μm (A), 20 μm (B–D). oped spinneret; terminal region A New Genus of Draconematids from Korea 79

arranged in two sublateral longitudinal rows; each row consisting of two adhesion tubes and no intermingling somatic setae. Anterior SlAT located at 74.9% of body length from anterior end, slightly longer than posterior SlAT, 51 and 45 μm long, respectively; distance of between two SlAT 6.2% of body length. Genital primordium 31 μm in length and consisting of a few cells. Tail with non-annulated but finely punctuated terminus, 55.1% of total tail length. Short anal flap present; not crenate. Sexual dimorphism. Males and females are very similar in most respects, other than the shape of the mid-body region (body diameter larger in female because of well- developed female reproductive system), and the ratio of the tail length to the non-annulated tail terminus (1.7 in female vs 2.2 in male). Females have a much shorter tail, but an equally long non-annulated terminal part.

Phylogenetic analysis of molecular data and genetic distances Nearly complete 18S ribosomal RNA gene sequences were determined for 10 draconematid nematodes and one epsilonematid nematode for the first time in the present study: Draconema japonicum (1,749 nucleotides), Megadraconema cornutum gen. nov., sp. nov. (1,691 nucleotides), Paradraconema sp. (1,691 nucleotides), Paradraconema jejuense (1,690 nucleotides), Dracograllus sp. 1 (1,690 nucleotides), Dracograllus sp. 2 (1,752 nucleotides), Prochaetosoma sp. 1 (1,689 nucleotides), Prochaetosoma sp. 2 (1,690 nucleotides), Prochaetosoma sp. 3 (1,689 nucleotides), Prochaetosoma sp. 4 (1,689 nucleotides) and Epsilonema sp. (1,690 nucleotides). A multiple alignment analysis of sequences from 12 nematodes including two outgroup taxa showed 1,768 positions including indels (insertions and/or deletions). Of these, our phylogenetic analyses were carried out based on the unam- biguously aligned regions including a total of 1,661 nucleotide positions with gaps included. The aligned sequences included 213 (12.8%) variable characters and 102 (6.1%) parsimony-informative characters. The A + T content of Megadraconema cornutum gen. nov., sp. nov. was 52%. Fig. 8. Megadraconema cornutum gen. nov., sp. nov., DIC photo- Tree topologies generated by the MP, ML and BI were micrographs of juveniles (A–C, lateral view) and SEM photomicro- exactly congruent with each other, and are represented by graph of juvenile (D, en face view). (A) Habitus, fourth-stage juvenile the maximum likelihood tree in Fig. 10. The Bayesian infer- (paratype RIT755). (B) Habitus, third-stage juvenile (paratype ence showed a higher degree of supporting values than ML RIT756). (C) Habitus, second-stage juvenile (paratype RIT757). (D) and MP analyses, except for the clade consisting of Head region, fourth-stage juvenile (paratype KORDI025), showing Prochaetosoma sp. 3 and P. sp. 4. The clade representing protruding lips, a transverse circle of cuticular papillae-like protru- the subfamily Prochaetosomatinae, including only four spe- sions and four cephalic adhesion tubes. Scale bars: 100 μm (A–C), 15 μm (D). cies of the genus Prochaetosoma, always formed a single monophyletic group with high support values (> 82%). The monophyletic Prochaetosoma was divided into two clades non-annulated but finely punctated, 49.5% of total tail with relatively low support values: ((P. sp. 1 + P. sp. 2) + (P. length. Anal flap short, not crenate. sp. 3 + P. sp. 4)). The subfamily Draconematinae is shown to Second-stage juvenile (paratype RIT757). Habitus sim- be paraphyletic, because the genus Dracograllus (D. sp. 1 ilar to adults (Figs. 8C, 9C). Body with strongly swollen pha- and D. sp. 2) did not group with the other members of the sub- ryngeal region, 11.3% of total body length. Head capsule family (Draconema, Megadraconema and Paradraconema) with a transverse circle of papillae-like protrusions (Fig. 9E); in any of our analyses. The genus Dracograllus diverged protrusions absent in molting second-stage juvenile (Fig. first at the basal line of all analyzed draconematid nema- 9D). Shape of amphidial fovea differing from adults, third todes, with very high supporting values in all analyses and fourth stage juveniles: a pore-like opening connected to (100%). In all analyses Megadraconema cornutum gen. an elongated spiral fovea, 21.1% of head capsule length. nov., sp. nov. clustered with a group including One CAT located dorsally about mid head capsule. PAT Paradraconema + Draconema, but the supporting values 80 H. S. Rho et al.

todes, based on the p-distance method using MEGA ver.4.0 to test the potential of 18S rRNA gene sequences as a molecular marker for species recognition and discrimination (Tables 5 and 6). The results of this analysis showed different rates of evolutionary substitution among taxa. Representatives of the family Epsilonematidae, used as outgroup taxa, showed relatively higher genetic distances than the species of the family Draconemati- dae as shown in Table 5 and 6. In the genus Paradraconema, the percentage of sequence distance and total nucleotide difference between two congeneric species, P. sp. and P. jejuense, were 2% and 31 bp. In the genus Prochaetosoma, the percentage of sequence distance and the total nucleotide difference among four congeneric species were very low and ranged from 0.3 to 0.7% and from 4 to 11 bp, respectively. The percentage of sequence distance and total nucleotide difference between two congeneric species of the genus Dracograllus, D. sp. 1 and D. sp. 2, were 2.8% and 44 bp, which is the highest value of genetic distance among species of Draconematidae examined in this study (Table 5). The mean distance of 18S rRNA gene sequences among genera within the family Draconematidae ranged from 1.4 (between Megadraconema and Prochaetosoma) to 3.6% (Paradra- conema and Dracograllus), and congeneric species differed by from 0.5 (Prochaetosoma) to 2.7% (Dracograllus) (Table 6). DISCUSSION Relationships within the family based on morphological features Fig. 9. Megadraconema cornutum gen. nov., sp. nov., third-stage juvenile (paratype RIT756, The first phylogenetic analysis A, B) and second-stage juvenile (paratype RIT757, C, D; KORDI022, E). (A) Habitus, lateral of the family Draconematidae was view. (B) Head region, external view. (C) Habitus, lateral view. (D) Head region, external view. based on 13 apomorphic morpho- (E) Head capsule with papillae-like protrusions. Scale bars: 50 μm (A, C), 10 μm (B, D, E). logical characters for 14 genera (Decraemer et al., 1997). The strict were very low and variable (20–67%). The two consensus tree based on maximum parsimony in this study Paradraconema species, P. sp. and P. jejuense clustered suggested two monophyletic groups and six genera of the together, with 53–98% as supporting values, and then also Prochaetosomatinae with unresolved position (Decraemer grouped with D. japonicum with relatively low supporting et al., 1997). Of the two monophyletic groups, one group values (26–63%). was formed by the genera of the subfamily Draconematinae In this study, we also analyzed the genetic distance of (Dracograllus Allen and Noffsinger, 1978, Draconema 18S rRNA gene sequences for 12 nematode species, Cobb, 1913, Paradraconema Allen and Noffsinger, 1978), including 10 draconematid and two epsilonematid nema- including Tenuidraconema Decraemer, 1989 (previously A New Genus of Draconematids from Korea 81 classified in Prochaetosomatinae) and one group formed by these clades remained unresolved. The Draconematinae is the solely deep-sea genera (Bathychaetosoma Decraemer characterized by three synapomorphies: (1) general body et al., 1997, Cephalochaetosoma Kito, 1983 and Dinetia shape with pronounced enlarged pharyngeal region, (2) Decraemer and Gourbault, 1997). The interrelationships within dumbbell-shaped pharynx and (3) narrow and unarmed buccal cavity. Tenuidraconema are considered as closely related with the three genera of the Draconematinae since its differing structure, cylindrical pharynx with well-developed end bulb, is considered to be due to a secondary loss of the dumbbell-shaped pharynx. The new genus Megadraconema gen. nov. possesses the three putative synapomorphies of the subfamily Draconematinae and is hereby assigned to it. Megadraconema cornutum gen. nov., sp. nov. differs from the other genera of the subfamily by the presence of a pore-like amphidial opening and an inner tubular fovea (not visible with SEM), the presence of a transverse circle of well- developed papilla-like cuticular protrusions and reticulate appearance of the cephalic subcuticle. The structure of the amphid with pore-like opening and inner tubular fovea as well as the dimorphism in shape of the amphidial fovea in second stage juveniles, differs from what was found in all other taxa of the family Dra- conematidae. The small, bar-shaped fovea observed in Dracognomus is different, as it is formed by an excavation of the cuticle and thus visible in SEM (Karssen and van Aelst, 2002). Megadraconema cornutum gen. nov., sp. nov. is considered most closely related to species of the genus Fig. 10. Phylogenetic tree of the family Draconematidae based on an alignment of 18S rRNA gene sequences recovered for maximum likelihood. Congruent topologies were Draconema in the general habitus and in recovered using Bayesian inference, maximum likelihood, and maximum parsimony. head structure, but can be easily distin- Two epsilonematid nematodes, Epsilonema sp. and Epsilonematidae sp., were desig- guished from that genus by the character nated as outgroup taxa. Numbers indicated above each internode are the bootstrap val- states mentioned above. SEM photos of ues for the maximum likelihood; numbers below are supporting values (posterior Draconema japonicum (see Rho and probabilities) for Bayesian inference (top), and bootstrap values for the maximum parsi- Kim, 2004b: 243, Fig. 8A) also show six mony (below). protruded lips, each with a setiform sen-

Table 5. Pairwise genetic distance based on p-distance method of 18S rRNA gene sequence of the family Draconematidae and Epsilone- matidae used in this study (above diagonal: number of differences; below diagonal: pairwise distance).

Taxa 123456789101112 1. Draconema japonicum – 293633614927253025207120 2. Megadraconema cornutum gen. nov., sp. nov. 0.019 – 36 31 59 52 22 20 27 22 204 120 3. Paradraconema sp. 0.023 0.023 – 31 65 60 32 32 33 30 213 126 4. Paradraconema jejuense 0.021 0.02 0.02 – 59 51 25 25 32 27 209 113 5. Dracograllus sp. 1 0.039 0.037 0.042 0.038 – 44 51 49 56 51 209 134 6. Dracograllus sp. 2 0.031 0.033 0.038 0.033 0.028 – 46 46 51 46 210 123 7. Prochaetosoma sp. 1 0.017 0.014 0.02 0.016 0.033 0.029 – 4 11 6 212 116 8. Prochaetosoma sp. 2 0.016 0.013 0.02 0.016 0.031 0.029 0.003 – 11 6 210 116 9. Prochaetosoma sp. 3 0.019 0.017 0.021 0.02 0.036 0.033 0.007 0.007 – 9 214 120 10. Prochaetosoma sp. 4 0.016 0.014 0.019 0.017 0.033 0.029 0.004 0.004 0.006 – 210 116 11. Epsilonema sp. 0.132 0.13 0.136 0.133 0.133 0.134 0.135 0.134 0.137 0.134 – 201 12. Epsilonematidae sp. 0.077 0.077 0.08 0.072 0.086 0.079 0.074 0.074 0.077 0.074 0.128 – 82 H. S. Rho et al.

Table 6. Mean % sequence distances within and between differ- A study of the other genera of the Prochaetosomatinae as ent nematode taxa. well as a more highly evolving gene sampling are necessary for a more reliable investigation of the draconematid rela- Taxa % sequence distances tionships. Within Draconematidae 2.2 Several attempts to develop molecular diagnostic tech- Within Epsilonematidae 12.9 niques using 18S rRNA gene sequences have been Within Dracograllus 2.7 achieved among various nematode taxa, such as plant par- Within Paradraconema 2 asitic nematodes, soil nematodes, and free-living marine Within Prochaetosoma 0.5 nematodes (Floyd et al., 2002, 2005; Foucher and Wilson, Draconematidae-Epsilonematidae 10.5 2002; Waite et al., 2003; Power, 2004; Cook et al., 2005; Megadraconema-Draconema 2 Bhadury et al., 2006a, b, 2007, 2008). Holterman et al. Megadraconema-Paradarconema 2.1 (2006) also mentioned that although a defined number of Megadraconema-Dracograllus 3.4 nucleotide differences cannot always be linked unequivo- Megadraconema-Prochaetosoma 1.4 cally, nematodes with relatively high substitution rates can Draconema-Paradraconema 2.3 mostly be recognized using a DNA barcode-based identifi- Draconema-Dracograllus 3.4 cation. The genetic distance of 18S rRNA gene sequences Draconema-Prochaetosoma 1.8 for marine draconematid and epsilonematid nematodes are Paradraconema-Dracograllus 3.6 so far unknown. According to our findings, based on the Paradraconema-Prochaetosoma 1.8 mean genetic distances of 18S rRNA sequences shown in Dracograllus-Prochaetosoma 3 Table 6, the draconematid nematodes have relatively lower genetic distances than the epsilonematid nematodes (2.2% sillum and a thin-walled collar-like part between the lips and vs 12.9%). Dracograllus showed the fastest substitution the thickened head capsule. rates (2.7%), and Prochaetosoma showed the slowest substitution rates (0.5%) within the Draconematidae. Moreover, Phylogenetic relationships within the family based on the mean genetic distance of 18S rRNA gene sequence molecular analyses among genera within the family Draconematidae ranged Our molecular phylogenetic analysis is largely in concor- from 1.4 to 3.6% and congeneric species differed by from dance with the results of the previous phylogenetic analysis 0.5 to 2.7%. On the basis of the above results, we recog- of Decraemer et al. (1997) based on morphological synapo- nized that the genetic distance of 18S rRNA gene sequence morphies except for the phylogenetic position of the genus has a possibility for the recognition and discrimination Dracograllus. The cluster of the two species of Dracograllus among the marine draconematid and epsilonematid nema- was not consistently grouped with the other members of the todes. subfamily Draconematinae: it diverged earlier, at the root of DICHOTOMOUS KEY TO GENERA OF THE FAMILY all draconematids used in this study; the node is supported by high bootstrap support values (100%) in all analyses (Fig. DRACONEMATIDAE 10). The branching order of the draconematid nematodes was The following dichotomous key to genera of the family as follows: (Dracograllus ((Megadraconema (Draconema, Draconematidae was developed based on the key charac- Paradraconema)) Prochaetosoma)). In the previous morpho- ters of the previous literatures by Allen and Noffsinger (1978) logical analyses of Decraemer et al. (1997), Dracograllus and Decraemer et al. (1997), and we just added the new was grouped with the other members of the subfamily apomorphic character of the new genus, Megadraconema Draconematinae, and the clade was supported by the pres- gen. nov., such as the structure of the subcuticle of head ence of a dumbbell-shaped pharyngeal apparatus and region and a transverse circle of well-developed papillae-like narrow, unarmed buccal cavity. This hypothesis is not in protrusions near anterior end of head capsule. concordance with present results based on 18S rRNA gene sequences, and indicates that the synapomorphic morpho- 1. Head capsule present ...... 2 logical characters uniting the members of the subfamily – Head capsule absent ...... Draconematinae need to be reconsidered and checked for ...... Dinetia Decraemer and Gourbault, 1997 possible convergent evolution especially with relation to 2(1). Pharynx with swollen corpus and swollen posterior Dracograllus. According to the putative synapomorphic bulb (dumbbell-shaped)...... 3 morphological conditions proposed by Decraemer et al.’s – Pharynx with cylindrical corpus, or with minor swelling, (1997) phylogenetic analysis, Megadraconema cornutum and terminal posterior bulb ...... 6 gen. nov., sp. nov. appears to be closely related to the sub- 3(2). Cephalic sublateral acanthiform setae present on head family Draconematinae. Our results, generated from the 18S capsule ...... Paradraconema Allen and Noffsinger, 1978 rRNA gene sequences, support a similar relationship of – Cephalic sublateral acanthiform setae absent on head Megadraconema cornutum gen. nov., sp. nov. with its basal capsule (except in Dracograllus steckhoveni)...... 4 position within the subfamily Draconematinae compared to 4(3). Swollen anterior body region less than 13% of total the constituents of Paradraconema and Draconema, but the body length...... 5 supporting values were very low and variable depending on – Swollen anterior body region more than 18% of total body the analyses (20–67%) (Fig. 10). We also have to take into length (18–25%) ... Dracograllus Allen and Noffsinger, 1978 account that in the present study only four species of the 5(4). Anteriormost annules (7–16) larger with subcuticular genus Prochaetosoma represent the Prochaetosomatinae. granulation ...... Draconema Cobb, 1913 A New Genus of Draconematids from Korea 83

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Mol Biol Evol 23: 1792–1800 thanks the Director General Dr. Pisani of the Royal Belgian Institute Hwang UW, Choi EW, Kim DS, Decraemer W, Chang CY (2009) of Natural Sciences for the kind hospitality and assistance. The Monophyly of the family Desmoscolecidae (Nematoda, authors thank “KUE (Korea Underwater Explorer)” members and Desmoscolecida) and its phylogenetic position inferred from Jae Wan Choi for their assistance in collecting samples. This work 18S rDNA sequences. Mol Cells 27: 515–523 was supported by the Korea Research Foundation Grant funded by Karssen G, van Aelst AC (2002) SEM observations on the marine the Korean Government (MOEHRD) to Hyun Soo Rho (KRF-2007- nematode Dracognomus simplex (Gerlach, 1954) Allen & 357-C00105) and also has been conducted with the support Noffsinger, 1978 (Draconematidae: Prochaetosomatinae). J through the research programme of KORDI with contract Nos. Nematol 34: 303–311 PE98444 and PE9842B to Hyun Soo Rho. 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